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Pravastatin-loaded Chitosan/Gelatin Composite Vascular Tissue Engineering Scaffold

Posted on:2016-06-17Degree:MasterType:Thesis
Country:ChinaCandidate:J FengFull Text:PDF
GTID:2284330482456687Subject:Pharmacy
Abstract/Summary:PDF Full Text Request
Cardiovascular disease have become the leading cause of death and affect humans life seriously. Vascular transplantation are usually used for patients with severe illness. The fundamental requirement for vascular grafts are biocompatibility, non-thrombogenicity and mechanical strength. There are several vascular grafts used clinically, including autologous vessels, allograft vessels and artificial blood vessel. Autologous grafts are native and biocompatible to the patients, thus can avoid the risk of immune rejection. However, its inadequate source limited its clinical use. Allografts also cannot be used widely because of the severe immunological rejection and desquamated cells in the earlier period after transplantation. Also, there are some ethical issues when using allografts and xeografts. Artificial blood vessel, which are made of expanded-polytetrafluoroethylene (ePTFE), dracron or polyurethance in the earlier stage, are not suitable for small-diameter vessel. Bessides the artificial vessels are not prone to thromboe mbolish, angiostenosis, calcium deposition, ect, and have no biological activity and reconstruct ability.The concept of the tissue engineering provides a new way for the vessel repair, in which the cells and biodegradable scaffold are co-cultured, then the cell-material composite is transplanted into the body. With the degradation of scaffold and the proliferation of the cells, a new vessel with physiological fuctions can be reconstructed. In order to promote the repair of damaged oragans, the researcher are mostly developed to how to acceletrate tissue growth. However, cardiovascular disease has the characteristics of high recurrence, and is often accomopanied with surgical complications. So how to repair the vascular lesion and prevent recurrence is the aim of our study.Statins, which competitively inhibit 3-hydroxy-3-methyglutarylcoenzyme (HMF-CoA) reductase, can repair the injured vascular endothelium and delay the development of atheromatous plaque along with the capability of anti-inflammation、 anticoagulation and hypolipidemic effect. Therefore statins can be used to prevente restenosis and reduce the surgical complications clinically. Pravastatin is a kind of water-soluble statin with less adverse reaction, having pharmacological activity in vivo without metabolic transformation, Currently, pravastatin is mainly by oral administration, which shows a short half-life and low bioavailability. Moreover, pravastatin is unstable in gastric acid environment, and easily converted to the isomer which possesses only 1/10-1/40 pharmacological activity of pravasttin.In this work, drug delivery system (DDS) was introduced into fabrication of tissue engineering scaffold. Chitosan microspheres with long-term release of pravastain sodium were prepared, then loaded into gelatin/itosan three-dimensional porous scaffold, aiming to develop a vascular tissue engineering scaffold which can reconstruct impaired blood vessel lesion, and prevent the recurrence, reduce the surgical complications meanwhile. Genipin, a natural and non-toxic cross-linking reagent, which can spontaneously react with chitosan or gelatin to form stable products, was used as cross-linker in our study.Genipin cross-linked chitosan microspheres loading pravastatin sodium for sustained release system were prepared by emulsion-cross-linking method. The influences of molecular weight of chitosan, volume ratio of oil and water, reaction temperature, stirring speed, the way of adding the crosslinkng agent, the concentration of genipin and the crosslinking time on the formation of chitosan microspheres after the processes of purifying the chitosan were investigated. The optimal formulation was obtained with chitosan (viscosity:200-400mpa.s), genipin 13g/L of polymer, reaction temperature of 40℃, stirring rate of 850 r/min, oil-water proportion of 10:1 and adding the genipin by twice about 25-30min each time.The crosslinking reaction was allowed to proceed for different time periods.The hardened microspheres were then separated by centrifugation and washed three times with petroleum ether, isopropyl and acetone by centrifugation process respectively. The prepared microspheres were vacuum dried at room temperature.SEM indicated the optimized microspheres are characterized by fine homogeneity and small size.We can conclude that prolonging the crosslinking time can help to further soothe surface of the microspheres and keep their dispersibility. Inappropriate concertration of cross-linking agent would create a bad influence on the shape of the microspheres. The swelling of chitosan microsphere prepared with different crosslinking time and different concentration of genipin were measured.The swelling response of microsphere exposed to media of different pH was also measured.The chitosan microsphere swelled most in acidic medium compared to the neutral or basic.The swelling ratio of the microspheres crosslinked for 4h were 237-332.4% when the microsphere were exposed to a media of pH ranged 2 from 10.Malvrn laser particle size analyzer revealed that the diameter or size distribution of the pravastatin loaded microsphere was hardly affected when compared with blank microsphere. FTIR confirmed cross-linked of genipin with chitosan and the absence of chemical interactions between drug, chitosan and cross-linker. Our results revealed that, chitosan have been successfully loaded with pravastatin sodium and the entrapment efficiency is 54.7%.Varying crosslinking time from 6-48h with 13g/L of genipin resulted decrease in drug release from the microspheres accordingly.The release from the microspheres were anged 30.3 from 6.3%.The microspheres crosslinked with increasing concentration of genipin (1.3,2.6,6.5and13g/Lwt% for crosslinking time 24h) decreased the release from 26.9 microspheres showed a sustained controlled release of the drug and the initial fast release phase was not observed. It can be seen from the SEM photos microsphere incubated for 105 days, some holes resulted from erosion appeared, which suggests that the chitosan microspheres has begun to degrade, thus the drug release futther; while in the early stage, pravastatin release should be due to the swelling of microsphere.Chitosan and gelatin based scaffolds can be considered as potential candidates for vascular tissue regeneration due to controllable mechanical property. Here, the study was conducted to investigate the possibility of using different concentration of genipin and EDC/NHS as the green cross-linker to modify chitosan/gelatin scaffolds for vascular tissue applications by freeze-drying method. The microstructure, porosity, enzyme degradation rate, and drug release from scaffold were investigated in details. The scaffold crosslinked with genipin showed better performance. There were appropriate pore size and fine morphology of chitosan40/gelatin60 scaffolds. The poroisity were ranged from 81.9%-96.1%. Increasing the concentration of cross-linking agent seemed to have a slight influence on the porosity of the scaffold. The scaffold crosslinked with genipin obtain more porosity than cross-linked with EDC/NHS. Besides, the scaffold cross-linked with genipin have slower degradation than cross-linked with EDC/NHS in lysozyme solution.The results revealed that scaffold crosslinked with geninp show more potential for preparing the vascular scaffold.Only 4.1%-24.6% pavastatin sodium was released from the microsphere within 40 days.The microspheres cross-linked with increasing concentration of genipin decreased the release.It can be inferred that sustained drug release was achieved by regulating the cross-linking time. In conclusion, pravastatin loaded chitosan/gelatin scaffold have obviously sustained effect.Cytotoxicity experiments were analyzed via-(4.5-Dimethylthiazol-2-ly)-2,5-diphenyl tetrazolium bromide(MTT) assay after 2 days by culturing for the human unmbilical vein endothelial cells (HUVECs)with the extract of the scaffolds. The result indicated that all the scaffolds are of ranking I and qualified, according to the evaluation criterion in ISO10933. The viability of the human unmbilical vein endothelial cells seeded on to the scaffold as assessed by cell counting.The result showed that drug release from the scaffold didn’t influence the cell growth in a short period time.
Keywords/Search Tags:chitosan, gelatin, tissue engineered blood vascular scaffold, pravastatin, microspheres, controlled-release
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